Therapeutic oligonucleotides, such as antisense oligonucleotides or ribozymes, are short segments of DNA that have been designed to hybridize to a sequence on a specific mRNA. The resulting complex can down-regulate protein production by several mechanisms, including inhibition of mRNA translation into protein and/or by enhancement of RNase H degradation of the mRNA transcripts. Consequently, therapeutic oligonucleotides have tremendous potential for specificity of action (i.e. the down-regulation of a specific disease-related protein). To date, these compounds have shown promise in several in vitro and in vivo models, including models of inflammatory disease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech. 14:376-387 (1996)). Antisense can also effect cellular activity by hybridizing specifically with chromosomal DNA. Advanced human clinical assessments of several antisense drugs are currently underway. Targets for these drugs include the genes or RNA products of c-myc, ICAM-1, and infectious disease organisms such as cytomegalovirus, and HIV-1.
One well known problem with the use of therapeutic oligonucleotides having a phosphodiester internucleotide linkage is its very short half-life in the presence of serum or within cells. (Zelphati, O et al. 1993. Inhibition of HIV-1 Replication in Cultured Cells with Antisense Oligonucleotides Encapsulated in Immunoliposomes. Antisense. Res. Dev. 3:323-338; and Thierry, AR et al. pp147-161 in Gene Regulation: Biology of Antisense RNA and DNA (Eds. Erickson, RP and Izant, JG) 1992. Raven Press, NY). No clinical assessment currently employs the basic phosphodiester chemistry found in natural nucleic acids, because of these and other known problems.
This problem has been partially overcome by chemical modifications which reduce serum or intracellular degradation. Modifications have been tested at the internucleotide phosphodiester bridge (i.e. using phosphorothioate, methylphosphonate or phosphoramidate linkages), at the nucleotide base (i.e.5-propynyl-pyrimidines), or at the sugar (i.e. 2'-modified sugars) (Uhlmann E., et al. 1997. Antisense: Chemical Modifications. Encyclopedia of Cancer Vol. X. pp 64-81 Academic Press Inc.). Others have attempted to improve stability using 2'-5' sugar linkages (see U.S. Pat. No. 5,532,130). Other changes have been attempted. However, none of these solutions have proven entirely satisfactory, and in vivo free antisense still has only limited efficacy. Problems remain, such as in the limited ability of some antisense to cross cellular membranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082 (1994)) and in the problems associated with systemic toxicity, such as complement-mediated anaphylaxis, altered coagulatory properties, and cytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206 (1994)). Further, as disclosed in U.S. patent application Ser. No. 08/657,753 and counterpart patent application WO 97/46671, both incorporated herein by reference, modified antisense is still highly charged, and clearance from the circulation still takes place within minutes.
To attempt to improve efficacy, investigators have also employed lipid-based carrier systems to deliver chemically modified or unmodified antisense. In Zelphati, O and Szoka, F. C. (1996) J. Contr. Rel. 41:99-119, the authors refer to the use of anionic (conventional) liposomes, pH sensitive liposomes, immunoliposomes, fusogenic liposomes and cationic lipid/antisense aggregates.
None of these compositions successfully deliver phosphodiester antisense for in vivo therapy. In another paper, Zelphati & Szoka note that antisense phosphodiester oligonucleotides associated with cationic lipids have not been active in cell culture in vitro; and that only one study has reported the activity of phosphodiester antisense oligonucleotides complexed to cationic lipids. The authors argue that these findings " . . . necessitate[ ] the use [of -sic] backbone-modified oligonucleotides that are relatively resistant to both intracellular and extracellular nucleases even if a carrier is used to deliver the oligonucleotide into the target cell". (1997. J. Lip. Res. 7(1):31-49 at 34). This finding is corroborated by Bennett, C F. (1995. Intracellular Delivery of Oligonucleotides with Cationic Liposomes. Chp 14 CRC Press) who states at p. 224 that "In contrast, we have been unable to demonstrate inhibition of gene expression by uniform phosphodiester oligodeoxynucleotides directed towards a number of cellular targets in the presence of cationic lipids."
Prior art lipid formulations of modified antisense are also largely ineffective in vivo. They have poor encapsulation efficiency (15% or less for passive encapsulation systems), poor drug to lipid ratios (3% or less by weight), high susceptibility to serum nucleases and rapid clearance from circulation (particularly in the case of cationic lipid/antisense aggregates made from DOTMA, trade-name LIPOFECTIN.TM.), and/or large sized particles (greater than 100 nm), which make them unsuitable for systemic delivery to target sites. No successful in vivo efficacy studies of lipid-encapsulated (nuclease-resistant) modified antisense are known in the prior art.
Two references to unique lipid-antisense compositions that may be significantly nuclease resistant bear consideration. Firstly, the anionic liposome (LPDII) composition of Li, S. and Huang, L (1997. J. Lip. Res. 7(1) 63-75), which encapsulates poly-lysine coated antisense, are said to have 60-70% encapsulation efficiency, but suffer from a large size of around 200 nm and a low drug to lipid ratio of 8% by weight. The effect of these particles in vivo is unknown. Secondly, the Minimal Volume Entrapment (MVE) technique for cardiolipin (anionic) liposomes results in the reasonably high encapsulation efficiency of 45-65% but again the drug:lipid ratio remains very small, approximately 6.5% by weight (see U.S. Pat. No. 5,665,710 to Rahman et al.; Thierry A R, and Takle, G B. 1995, Liposomes as a Delivery System for Antisense and Ribozyme Compounds. in Delivery Strategies for Antisense Oligonucleotide Therapeutics, S. Akhtar, ed, CRC Press, Boca Raton, Fla., pp. 199-221; Thierry, A R et al. pp147-161 in Gene Regulation: Biology of Antisense RNA and DNA (Eds. Erickson, R P and Izant, J G) 1992. Raven Press, NY). Note that U.S. Pat. No. 5,665,710 also discloses encapsulation efficiencies of 60-90% for tiny, medically useless amounts of antisense (0.1 ug), where the drug to lipid ratio must be very low.
It is an observation of the inventors that a wide variety of prior art lipid compositions used for conventional drugs could be tested for efficacy in the antisense field, but the improvement (over free antisense) for in vivo efficacy is not known. In this regard, it is noted that although lipid compositions assertedly for use as drug carriers were disclosed by Bailey and Cullis (U.S. Pat. No. 5,552,155; and (1994) Biochem. 33(42):12573-12580), they did not disclose formulations of any bioactive compounds with these lipids, and did not suggest their utility for high efficiency loading of polyanionic species.
What is needed in the art are improved lipid-therapeutic oligonucleotide compositions which are suitable for therapeutic use. Preferably these compositions would encapsulate nucleic acids with high-efficiency, have high drug:lipid ratios, be encapsulated and protected from degradation and clearance in serum, and/or be suitable for systemic delivery. The present invention provides such compositions, methods of making the compositions and methods of introducing nucleic acids into cells using the compositions and methods of treating diseases.